Production of biologically active thymulin in Escherichia coli through expression of a chemically synthesized gene

Summary A synthetic gene coding for thymulin was ligated into an expression vector (pJB 1301) and placed under lac operon control. In the recombinant clones, thymulin was expressed as part of a galactosidase chimeric protein which was then cleaved by cyanogen bromide. Thymulin was purified using various chromatography systems including gel filtration and HPLC, and was detected by radioimmunoassay (RIA). In the biological assay the purified recombinant peptide demonstrated the same zinc dependency as natural thymulin and had the same amino acid composition and primary structure.     

Ecology and fish catches in natural lakes of Benin,West Africa

Synopsis A preliminary study of the ecology and commercial fish catches was carried out in the Toho-Todougba, Ahouangan and Dati lakes (1500 ha) in southern Benin (West Africa) over a period of 18 consecutive months. Water quality, species richness, and the reproductive biology of the dominant species, Sarotherodon galilaeus, were examined. Unlike Lagoon Toho-Todougba, lakes Ahouangan and Dati are less favourable for fish production because of their high total iron content and their relative low pH. The study revealed the existence of 19 species belonging to eleven (11) families: 16 species in Lagoon Toho-Todougba,12 in Lake Ahouangan, and 7 in Lake Dati. Lakes Ahouangan and Dati had a relatively high faunal similarity whereas Lagoon Toho-Todougba exhibited a relatively low faunal similarity with the former systems. In Lagoon Toho-Todougba, six cichlid species were the most important component of the fishery: Sarotherodon galilaeus, Tilapia guineensis, T. zillii, T. mariae, Chromidotilapia guntheri, Hemichromis fasciatus. Three catfish species Chrysichthys auratus, Clarias agboyiensis, and Clarias lazera were harvested in low abundances. Some other fish, such as Polypterus senegalus senegalus, Protopterus annectens and Heterotis niloticus were captured sporadically, with H. niloticus appearing more consistently in catches than the others. Although not abundant, Gymnarchus niloticus and the African pike, Hepsetus odoe, were almost always present in the catches. Ctenopoma kingsleyae appeared only in Ahouangan. Some predatory species, such as Notopterus afer and Xenomystus nigri, are encountered only in lakes Ahouangan and Dati. In Lagoon Toho-Todougba, Sarotherodon galilaeus had a sex ratio (: ) of 0.49:1, and size at maturation was estimated as 12.3 cm TL. The spawning period extended from May to September with the peak occurring in July. Fecundity was correlated to log-body length (r = 0.74) and log-body weight (r = 0.76). Fish catches were estimated at 389 kg ha^–1 year^–1; about 73% of which was Sarotherodon galilaeus. The overexploitation of juvenile fish and the use of this lagoon for the irrigation of palm tree plantations have reduced fish recruitment and mean size.     

Effects of medium and substratum conditions on the rates of DNA synthesis in primary cultures of bile ductular epithelial cells

Summary Select medium and substratum conditions were investigated for their effects on semiconservative DNA synthesis in essentially pure primary cultures of bile ductular epithelial cells that were initially isolated from cholestatic rat livers at 6 to 10 wk after bile duct ligation. DNA synthesis in these cultured cells was serum-dependent, being at its highest level when the concentration of fetal bovine serum present in the medium was maintained at 10%. This serum-dependent DNA synthesis was completely inhibited when 10 mM hydroxyurea was also included in the medium, and bile ductular cells cultured in the continued presence of 1.0% fetal bovine serum showed only marginal DNA synthesis during 8 to 10 d of primary culture when compared with no-serum controls. Maximum rates of serum-dependent DNA synthesis were obtained when the bile ductular cells were cultured for 7 to 14 d on tissue culture plastic coated with obtained when the bile ductular cells were cultured for 7 to 14 d on tissue culture plastic coated with either fibronectin from bovine plasma or type I rat-tail collagen. Cells cultured on plastic coated with basement membrane Matrigel exhibited the lowest levels of DNA synthesis, whereas those on plastic alone had intermediate amounts. Furthermore, the addition of epidermal growth factor (50 ng·ml⁻¹·d⁻¹) to medium supplemented with 1.0% fetal bovine serum greatly enhanced the rate of DNA synthesis in bile ductular cells after 6 d in primary culture on type I collagen-coated plastic over that measured in solvent control cultures. These findings indicate that our bile ductular epithelial cell culture model is potentially useful in the study of biliary cell growth regulation and carcinogenesis. This investigation was supported by USPHS grant RO1 CA 39225 to A. E. Sirica by the National Cancer Institute, Department of Health and Human Services, Bethesda, MD. During the period of this study, G. A. Mathis was a recipient of a Fellowship from the Fund for Academic Career Development of the State of Zurich, Switzerland.     

Regulation of the High Affinity IgE Receptor (FcεRI) in Human Neutrophils: Role of Seasonal Allergen Exposure and Th-2 Cytokines

The high affinity IgE receptor, FcεRI, plays a key role in the immunological pathways involved in allergic asthma. Previously we have demonstrated that human neutrophils isolated from allergic asthmatics express a functional FcεRI, and therefore it was of importance to examine the factors regulating its expression. In this study, we found that neutrophils from allergic asthmatics showed increased expression of FcεRI-α chain surface protein, total protein and mRNA compared with those from allergic non asthmatics and healthy donors (p<0.001). Interestingly, in neutrophils isolated from allergic asthmatics, FcεRI-α chain surface protein and mRNA expression were significantly greater during the pollen season than outside the pollen season (n = 9, P = 0.001), an effect which was not observed either in the allergic non asthmatic group or the healthy donors (p>0.05). Allergen exposure did not affect other surface markers of neutrophils such as CD16/FcγRIII or IL-17R. In contrast to stimulation with IgE, neutrophils incubated with TH2 cytokines IL-9, GM-CSF, and IL-4, showed enhanced FcεRI-α chain surface expression. In conclusion, these results suggest that enhanced FcεRI expression in human neutrophils from allergic asthmatics during the pollen season can make them more susceptible to the biological effects of IgE, providing a possible new mechanism by which neutrophils contribute to allergic asthma.     

Differential phosphorylation of tau proteins during kitten brain development and Alzheimer's disease

Differential distribution and phosphorylation of tau proteins were studied in developing kitten brain by using several antibodies, and was compared to phosphorylation in Alzheimer's disease. Several antibodies demonstrated the presence of phosphorylated tau proteins during kitten brain development and identified pathological structures in human brain tissue. Antibody AD2, recognized tau in kittens and adult cats, but reacted in Alzheimer's tissue only with a pathological tau form. Antibody AT8 was prominent in developing kitten neurons and was found in axons and dendrites. After the first postnatal month this phosphorylation type disappeared from axons. Furthermore, dephosphorylation of kitten tau with alkaline phosphatase abolished immunoreactivity of AT8, but not that of AD2, pointing to a protection of the AD2 epitope in cats. Tau proteins during early cat brain development are phosphorylated at several sites that are also phosphorylated in paired helical filaments during Alzheimer's disease. In either event, phosphorylation of tau may play a crucial role to modulate microtubule dynamics, contributing to increased microtubule instability and promoting growth of processes during neuronal development or changing dynamic properties of the cytoskeleton and contributing to the formation of pathological structures in neurodegenerative diseases.     

NMR solution structures of Runella slithyformis RNA 2′-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity

Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO₄ to NAD⁺ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD⁺. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD⁺-bound states. ITC measurements of RslTpt1 binding to NAD⁺ (K_D ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD⁺-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP⁺, which contains an adenylate 2′-PO₄ (mimicking the substrate RNA 2′-PO₄), binds with lower affinity (K_D ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD⁺ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD⁺ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.     

Independent and coordinated functions of replication protein A tandem high affinity single-stranded DNA binding domains

The initial high affinity binding of single-stranded DNA (ssDNA) by replication protein A (RPA) is involved in the tandem domains in the central region of the RPA70 subunit (RPA70AB). However, it was not clear whether the two domains, RPA70A and RPA70B, bind DNA simultaneously or sequentially. Here, using primarily heteronuclear NMR complemented by fluorescence spectroscopy, we have analyzed the binding characteristics of the individual RPA70A and RPA70B domains and compared them with the intact RPA70AB. NMR chemical shift comparisons confirmed that RPA70A and RPA70B tumble independently in solution in the absence of ssDNA. NMR chemical shift perturbations showed that all ssDNA oligomers bind to the same sites as observed in the x-ray crystal structure of RPA70AB complexed to d(C)8. Titrations using a variety of 5'-mer ssDNA oligomers showed that RPA70A has a 5-10-fold higher affinity for ssDNA than RPA70B. Detailed analysis of ssDNA binding to RPA70A revealed that all DNA sequences interact in a similar mode. Fluorescence binding measurements with a variety of 8-10'-mer DNA sequences showed that RPA70AB interacts with DNA with approximately 100-fold higher affinity than the isolated domains. Calculation of the theoretical "linkage effect" from the structure of RPA70AB suggests that the high overall affinity for ssDNA is a byproduct of the covalent attachment of the two domains via a short flexible tether, which increases the effective local concentration. Taken together, our data are consistent with a sequential model of DNA binding by RPA according to which RPA70A binds the majority of DNA first and subsequent loading of RPA70B domain is facilitated by the linkage effect.     

Rafts: a simple way to control apoptosis by subcellular redistribution

Apoptosis is an essential feature of development and homeostasis in higher organisms. Lipid rafts are subdomains of the plasma membrane that contain high concentrations of cholesterol and sphingolipids. In response to intra or extracellular stimuli, lipid rafts can include or exclude proteins to variable extents. This favors specific protein-protein interactions and modulates the activity of signaling cascades. Recently, a number of proteins involved in apoptotic signals have been located in lipid rafts. Among these proteins is included Bad, a pro-apoptotic molecule belonging to the Bcl-2 family. Bad is attached to lipid rafts in proliferating cells while associated to mitochondria in apoptotic cells, suggesting that the interaction of Bad with rafts is a dynamic process involved in the control of apoptosis. In this review, we briefly summarize the structure of rafts and illustrate their contribution to the control of apoptosis.     

The neuropathological spectrum of neurodegenerative tauopathies

Abundant neurofibrillary lesions made of abnormal and hyperphosphorylated microtubule-associated protein tau constitute one of the defining neuropathological features of Alzheimer's disease. However, tau containing filamentous deposits in neurons and/or glial cells also define a heterogeneous group of neurodegenerative disorders clinically characterized by dementia and/or motor syndromes. Thus, all these disorders are collectively grouped under the generic term of tauopathies. In the present review we outline the morphological and biochemical characteristics of some major tauopathies, including Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration and argyrophilic grain disease. The second part will deal with the recent discovery of tau gene mutations in frontotemporal dementia and parkinsonism linked to chromosome 17 which demonstrates that tau dysfunction can lead to neurodegeneration. Finally, we will discuss the very recent finding of 'tau-deficient' tauopathy in a subset of frontotemporal dementia cases.     

Oligomerization of acidic fibroblast growth factor is not a prerequisite for its cell proliferation activity

Oligomerization of fibroblast growth factors (FGFs) induced on binding to heparin or heparan sulfate proteoglycan is considered to be crucial for receptor activation and initiation of biological responses. To gain insight into the mechanism of activation of the receptor by FGFs, in the present study we investigate the effect(s) of interaction of a heparin analog, sucrose octasulfate (SOS), on the structure, stability, and biological activities of a recombinant acidic FGF from Notophthalmus viridescens (nFGF-1). SOS is found to bind to nFGF-1 and significantly increase the thermodynamic stability of the protein. Using a variety of techniques such as size-exclusion chromatography, sedimentation velocity, and multidimensional nuclear magnetic resonance (NMR) spectroscopy, it is shown that binding of SOS to nFGF-1 retains the protein in its monomeric state. In its monomeric state (complexed to SOS), n-FGF-1 shows significant cell proliferation activity. (15)N and (1)H chemical shift perturbation and the intermolecular nuclear Overhauser effects (NOEs) between SOS and nFGF-1 reveal that the ligand binds to the dense, positively charged cluster located in the groove enclosed by beta-strands 10 and 11. In addition, molecular modeling based on the NOEs observed for the SOS-nFGF-1 complex, indicates that SOS and heparin share a common binding site on the protein. In conclusion, the results of the present study clearly show that heparin-induced oligomerization of nFGF-1 is not mandatory for its cell proliferation activity.